Structural Analysis , mutation and therapeutic use of TCRs from HIV-specific CTLs

描述（由申请方提供）：HIV-1特异性CD 8细胞毒性T淋巴细胞（CTL）应答在控制HIV-1复制中起关键作用。TCR亲合力与CTL功能相关，并且表达对其同源MHC-病毒肽复合物具有高亲合力的TCR的CTL在中和病毒感染、终止病毒感染和延迟系统性AIDS病毒从粘膜接种部位传播中起重要的体内作用。关于HIV特异性CTL表达的TCR识别HIV肽：MHC I类复合物的结构基础以及特异性TCR残基对其同源HIV肽的TCR亲合力的贡献知之甚少。HIV特异性CD 8 + CTL表达的TCR识别HIV肽的结构-功能相关性的阐明将增加我们对一些TCR赋予CTL有效抗HIV活性的机制的理解。我们假设，在解析TCR结构并鉴定TCR-肽接触残基后，HIV特异性CTL的功能活性可通过接触同源肽的TCR氨基酸的突变而显著增强，从而增加其对其同源肽：MHC复合物的亲和力。我们已经将来自HIV特异性CTL克隆的TCR基因克隆到慢病毒载体中，所述慢病毒载体有效地将外周CD 8 T细胞转化为具有确定特异性的HIV特异性CTL。我们还将这些TCR基因作为连接的单链TCR（scTCR）克隆到细菌、果蝇和酵母载体中以表达足够的蛋白质来产生晶体以确定TCR结构。TCR对其同源HIV肽的亲合力的结构基础将通过确定由HLA-A*0201限制性HIV-SL 9特异性TCR及其同源I类MHC：SL 9-肽组装体形成的复合物的X射线晶体学结构来获得。这些结构将允许我们1）定义负责MHC的物理和化学决定因素：肽识别; 2）确定/合理化靶向突变对CTL功能的影响; 3）进行具有独特结合和生物活性的新型TCR的结构辅助设计。了解TCR识别HIV表位的结构基础可能有助于设计能够诱导CTL的疫苗，这些CTL以更高的亲合力识别HIV表位，并且在预防HIV感染方面更有效。我们最近报道了188 Re连接的HIV-1特异性人单克隆抗体可用于体内有效清除HIV-1感染细胞。我们将确定这种方法是否可以用于开发一种新的HIV治疗方法，该方法使用与放射性同位素偶联的可溶性TCR四聚体，无论是天然形式还是携带我们确定的旨在增加亲合力的突变，并检查其体外和体内靶向和消除HIV感染细胞的能力。 公共卫生相关性：我们建议通过解决由HIV-SL 9特异性TCR和同源MHC I类：SL 9-肽组装体形成的复合物的X射线晶体学结构来进行HIV特异性CTL的TCR的结构-功能分析。我们将定义负责MHC的物理和化学决定因素：肽识别，确定/合理化靶向突变对CTL功能的影响，并使用此信息作为基础的结构辅助设计的新的HIV治疗剂来自TCR连接到radiolefintides。

DESCRIPTION (provided by applicant): HIV-1 specific CD8 cytotoxic T lymphocyte (CTL) responses play a critical role in controlling HIV-1 replication. TCR avidity correlates with CTL function, and CTLs expressing TCRs with high avidity for their cognate MHC-viral peptide complex play an important in vivo role in neutralizing virus infections, terminating virus infection and delaying systemic AIDS virus dissemination from the mucosal inoculation site. Little is known about the structural basis by which the TCR's expressed by HIV-specific CTL recognize the HIV peptide:MHC Class I complex and the contributions of specific TCR residues to the TCR avidity for its cognate HIV peptide. Elucidation of the structure- function correlates of HIV peptide recognition by the TCR expressed by HIV-specific CD8+ CTLs will increase our understanding of the mechanism by which some TCRs confer CTLs with potent anti-HIV activity. We hypothesize that after solving the TCR structure and identifying TCR-peptide contact residues, the functional activity of HIV- specific CTLs can be markedly enhanced by mutation of TCR amino acids that contact the cognate peptide thereby increasing its affinity for its cognate peptide:MHC complex. We have cloned the TCR genes from an HIV-specific CTL clone into lentiviral vectors that efficiently transform peripheral CD8 T cells into HIV-specific CTLs with defined specificities. We have also cloned these TCR genes as a linked single chain TCR (scTCR) into bacterial, drosophila and yeast vectors to express sufficient protein to generate crystals to determine the TCR structure. The structural basis of TCR avidity for its cognate HIV peptide will be obtained by determining the X-ray crystallographic structures of the complexes formed by HLA-A*0201-restricted HIV-SL9-specific TCRs and their cognate MHC Class I:SL9-peptide assemblies. These structures will permit us to 1) define the physical and chemical determinants responsible for MHC:peptide recognition; 2) determine/rationalize the effect of targeted mutations on CTL function and 3) perform structure-assisted design of novel TCRs with unique binding and biological activities. Understanding the structural basis of TCR recognition of HIV epitopes may facilitate the design of vaccines capable of inducing CTLs that recognize HIV epitopes with a higher avidity and that are more effective in preventing HIV infection. We have recently reported that 188Re-linked HIV envelope-specific human mAb can be used to efficiently eliminate HIV-1-infected cells in vivo. We will determine if this approach could be used to develop a new HIV therapy using soluble TCR tetramers coupled to radioisotopes, either in the native form or carrying mutations we identified designed to increase avidity, and examine their in vitro and in vivo capacity to target and eliminate HIV-infected cells. PUBLIC HEALTH RELEVANCE: We propose to perform structure-function analysis of the TCR of HIV-specific CTLs by solving the X-ray crystallographic structures of the complexes formed by HIV-SL9- specific TCRs and cognate MHC Class I:SL9-peptide assemblies. We will define the physical and chemical determinants responsible for MHC:peptide recognition, determine/rationalize the effect of targeted mutations on CTL function and use this information as the basis for the structure-assisted design of novel HIV therapeutics derived from TCRs linked to radionucleotides.